This invention relates to a xcex2-primeverosidase of a plant. xcex2-Primeverosidase is an enzyme acting on xcex2-primeveroside, which is a disaccharide glycoside, and catalyzing a reaction of forming aroma components of tea and primeverose.
In the studies on aroma components of plants, there has been confirmed the presence of a disaccharide glycoside xcex2-primeveroside (6-O-xcex2-D-xylopyranosyl-xcex2-D-glucopyranoside) and its analogs, as precursors for alcoholic aroma components such as geraniol and linalol. Also, it has been clarified that xcex2-primeveroside, a disaccharide glycoside, and its analogs exist as precursors for alcoholic aroma components other than those cited above.
The invention relates to a xcex2-primeverosidase gene of a plant. xcex2-Primeverosidase is an enzyme acting on xcex2-primeveroside, which is a disaccharide glycoside, and catalyzing a reaction of forming aroma components of tea and primeverose.
The term xe2x80x9cxcex2-primeverosidasexe2x80x9d means an enzyme having an enzymatic activity of cleaving disaccharide glycosides (in particular, xcex2-primeveroside and its analogs) in a disaccharide unit. The enzyme according to the invention is characterized by having an activity of acting on a disaccharide glycoside, which is hardly usable as a substrate by the existing glucosidase, and thus releasing saccharides in a disaccharide unit from this disaccharide glycoside. An enzyme having the above activity is called xe2x80x9cxcex2-primeverosidasexe2x80x9d herein.
Concerning the presence of an enzyme specifically acting on these disaccharides, the inventors first studied xcex2-primeverosidase which is an enzyme forming the aroma components of tea. Thus, they isolated this enzyme and clarified its properties (JP-A-8-140675; the term xe2x80x9cJP-Axe2x80x9d as used herein means an xe2x80x9cunexamined published Japanese patent applicationxe2x80x9d).
However, the nucleotide sequence, etc. of this enzyme have not been clarified hitherto and no study has been made on the isolation and utilization of its gene.
An object of the invention is to provide means for isolating xcex2-primeverosidase gene originating in a plant, in particular tea, and thus providing this enzyme at a lower cost. The invention makes it possible to broaden the application range of the xcex2-primeverosidase originating in a plant.
To achieve the above-described object, the inventors have conducted intensive studies and, as a result, completed the invention.
Namely, the gist of the invention resides in a DNA which encodes a protein comprising the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing or an amino acid sequence derived from this sequence by deletion, substitution, insertion or addition of one or more amino acids.
Particular examples of the above-described DNA include the following DNAs. Namely, DNA having the nucleotide sequence represented by SEQ ID NO:2 in Sequence Listing, or a DNA as described in claim 1 which encodes a protein comprising a polynucleotide selected from the following polynucleotides (a) to (g) and has a primeverosidase activity:
(a) a polynucleotide encoding a polypeptide having the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing;
(b) a polynucleotide encoding a polypeptide having an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing by deletion, addition, insertion or substitution of one or more amino acid residues;
(c) a polynucleotide having the nucleotide sequence represented by SEQ ID NO:2 in Sequence Listing;
(d) a polynucleotide having a nucleotide sequence derived from the nucleotide sequence represented by SEQ ID NO:2 in Sequence Listing by deletion, addition, insertion or substitution of one or more bases;
(e) a gene capable of hybridizing with one of the polynucleotides (a) to (d) as described above under stringent conditions;
(f) a polynucleotide having a homology with one of the polynucleotides (a) to (d) as described above; and
(g) a polynucleotide which is degenerate with respect to at least one of the polynucleotides (a) to (f) as described above.
Now, the invention will be described in detail. The DNA according to the invention is a DNA encoding the above-described protein. To complete the invention, this DNA could be isolated from a tea cDNA library as will be described hereinafter. However, its nucleotide sequence has been clarified by the invention and, therefore, it can be obtained by chemical synthesis on the basis of the sequence represented by SEQ ID NO:1 or 2.
Alternatively, the DNA according to the invention can be obtained from a tea chromosomal DNA library or DNA libraries of other plants by the PCR or hybridization method known per se with the use of synthetic oligonucleotide probes or oligonucleotide primers synthesized on the basis of these sequences.
Next, illustration will be made by reference to examples of a method of obtaining the DNA according to the invention by acquiring a part of the DNA of the invention from a tea cDNA library by PCR followed by hybridization by using it as a probe, and a method of producing xcex2-primeverosidase from Escherichia coli or a yeast by a gene recombination method with the use of the thus obtained DNA.
It is widely recognized by those skilled in the art in general that an amino acid sequence encoding a physiologically active protein sometimes sustains its physiological activity in case of having deletion, substitution, insertion or addition of one or more amino acids. As a matter of course, the invention involves in the scope thereof DNA fragments having these modifications and yet encoding proteins having the xcex2-primeverosidase activity.
That is to say, the invention involves in the scope thereof DNAs encoding proteins which comprise amino acid sequences derived from the sequence represented by SEQ ID NO:1 in Sequence Listing by deletion, substitution, insertion or addition of one or more amino acids and have the xcex2-primeverosidase activity. Such a modified DNA can be obtained by modifying the nucleotide sequence of the invention so as to delete, substitute, insert or add amino acid(s) at specific site(s) by, for example, the site-directed mutagenesis method.
Alternatively, a modified DNA can be obtained by mutagenesis of the DNA according to the invention or cells having the same and selecting a DNA which is hybridizable with the DNA having, for example, the nucleotide sequence represented by SEQ ID NO:2 in Sequence Listing under stringent conditions from the DNAs or cells obtained above.
The term xe2x80x9cstringent conditionsxe2x80x9d as used herein means such conditions that allow the formation of so-called specific hybrids but not unspecific hybrids. Although these conditions can be hardly defined numerically, citation may be made of conditions under which nucleic acids having a high homology with each other (for example, DNAs having a 70 to 90% or more homology with each other) are hybridized but nucleic acids having a lower homology with each other cannot be hybridized.
The xe2x80x9cstringent conditionsxe2x80x9d may be exemplified by 6xc3x97SSC, 1.0% of a blocking agent, 0.1% of N-lauroylsarcocine sodium and 0.02% of SDS.
It is also possible to obtain a xcex2-primeverosidase gene from tea chromosome by a conventional method with the use of the DNA according to the invention or a part thereof as a probe. However, it is expected that the xcex2-primeverosidase gene originating in tea chromosome contains intron(s). Such a DNA having an intervening intron also falls within the scope of the DNA according to the invention, so long as it encodes the xcex2-primeverosidase of the invention.
Moreover, the invention involves in its scope a protein which comprises an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing by deletion, substitution, insertion or addition of one or more amino acids but has no xcex2-primeverosidase activity, so long as a protein having the xcex2-primeverosidase activity can be obtained therefrom by a simple treatment such as splicing or treating with a protease. Examples of such case include a protein having the amino acid sequence represented by SEQ ID NO:15 and proteins having amino acid sequences derived from the amino acid sequence represented by SEQ ID NO:15 by deletion, substitution, insertion or addition of one or more arbitrary amino acids in the region from the N-terminal xe2x88x9278 to xe2x88x921 positions.
Furthermore, the invention involves in its scope fused proteins composed of a protein having an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by deletion, substitution, insertion or addition of one or more amino acids with another protein. Examples thereof include proteins employed in fused protein expression systems (for example, maltose-binding protein: Pascale Duplay et al., Journal Biological Chemistry, Vol. 259, pp. 10606-10613 (1984)).
Further, a transformant capable of producing xcex2-primeverosidase can be selected by integrating the obtained xcex2-primeverosidase gene into an appropriate vector, transforming E. coli, yeasts, cultured cells, plants, etc. therewith, and then screening.
When cultured in a nutrient medium, the transformant thus obtained stably produces a peptide having a high xcex2-primeverosidase activity. The culture conditions in culturing the transformant may be appropriately selected by taking the nutritional and physiological properties of the host into consideration. Although liquid culture is usually carried out in many cases, submerged aeration-agitation culture is industrially advantageous. As a carbon source, any carbon compound may be used so long as it is metabolizable. For example, use can be made of glucose, lactose, maltose and the like. As a nitrogen source, any nitrogen compound may be used so long as it is usable. For example, use can be made of yeast extract, peptone, meat extract and the like. The culture temperature is controlled to such a level as allowing the production of xcex2-primeverosidase. In case of E.coli or yeasts, the culture temperature usually ranges from 10 to 42xc2x0 C. To express the xcex2-primeverosidase according to the invention in E.coli or yeasts, it is preferable to use a culture temperature of 20xc2x0 C. or lower, still preferably from about 10 to about 20xc2x0 C.
The culture may be carried out for a period of time of attaining the maximum yield of xcex2-primeverosidase. The culture is usually continued for 12 to 72 hours. The pH value of the medium may be controlled so as to allow the growth of the cells and stably produce xcex2-primeverosidase, preferably from pH 6 to 8.
Though the presence of the xcex2-primeverosidase gene originating in tea according to the invention had been clarified, it was highly difficult to confirm its activity. In the invention, it has been clarified that the expression of a mature protein as an active protein can be achieved by carrying out culturing under specific conditions by using a fused protein expression system.
That is to say, the expression could be first achieved by carrying out the culturing at 20xc2x0 C. or lower to thereby slow down the protein production speed compared with usual cases. In particular, expression in E.coli can be established by forming an inducible fused protein and thus slowing down the protein production. As the fused protein, it is favorable to use a protein fused with maltose-binding protein. It is also favorable that the production at a low speed is carried out by using promoters which can be artificially controlled (promoters lac, tac, etc.) and an inducer (for example, IPTG) at a lower concentration (1 mM or lower, preferably about 0.1 mM) than that in usual cases.
The xcex2-primeverosidase thus produced may be subjected to various treatments depending on the purpose. In case where the xcex2-primeverosidase is contained in cells, the cells are harvested by filtration, centrifugation, etc. and then disrupted by a physical method with the use of a machine or an enzymatic method with the use of lysozyme, etc. followed by extraction. Moreover, the xcex2-primeverosidase thus obtained may be salted out, concentrated, purified, etc., if needed.
Unless otherwise noted, the xcex2-primeverosidase activity described herein was measured in the following manner.
The enzyme activity was examined by using p-nitrophenyl-xcex2-primeveroside (pNP-Pri) as a substrate and measuring p-nitrophenol (pNP) released therefrom with a spectrophotometer. The unit of enzyme activity was defined as the release of 1 xcexcmol of pNP per minute.
More particularly, 300 xcexcl of a sample to be assayed, 1100 xcexcl of a 20 mM citrate buffer solution (pH 6.0) and 300 xcexcl of a 10 mM solution of pNP-Pri in the same buffer solution were mixed and incubated at 37xc2x0 C. After 30, 60, 90 and 120 minutes, the liquid reaction mixture was sampled in 340 xcexcl portions and the reaction was stopped by adding 170 xcexcl of a 1 M aqueous solution of sodium carbonate. Then the absorbance at 405 nm was measured with a spectrophotometer.
The pNP-primeveroside serving as the substrate can be synthesized by, for example, reacting pNP-glucoside (manufactured by Merck and Co., Inc.) with xylooligosaccharide (manufactured by Wako Pure Chemical Industries, Ltd.) by using an enzyme xylosidase (manufactured by Sigma Chemical Co.) and thus transferring one xylose residue to the pNP-glycoside via a xcex2-1,6 bond.
The enzyme activity was measured as in the measurement of the xcex2-primeverosidase activity (1) by using p-nitrophenyl-xcex2-glucoside as a substrate.
Now, the invention will be described in greater detail by reference to the following Examples. Needless to say, the invention is not construed as being limited thereto without departing from the gist of the invention. Unless other wise noted, all percentages are expressed in w/v % in this specification.